This experimental protocol describes several methods for generating complement-stabilized lentiviral vectors either by using fusion packaging proteins containing the complement regulatory protein CD55 (degradation acceleration factor, D A F ) to generate pseudotyped viral plasmids or by using naturally occurring D A F co-assembled with packaging proteins to generate novel lentiviruses.
Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Preparation of lentiviral vectors Move Preparation of lentiviral vectors Materials reagents CaCl2 (2mol/L) Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose (serum free complete) and 10 % fetal calf serum (FCS). 2 X HBS (HEPES-buffered saline) 50 mmol/L HEPES 280 mmol/L NaCl I. 5 mmol/L Na2 HP 〇 4 Adjust pH to 7 .12 with 5 mol/L NaOH. HEK 293 T Cells (ATCC, CRL 11268) Phosphate buffer P B S Plasmids DAF encoded transgenic plasmid Envelope-encoded plasmids (env) Gp64-DAF fusion protein encoding transgenic plasmid Packaging plasmids (pack) Transgenic plasmids VSV-ODAF fusion protein encoding transgenic plasmids 0.IX T E 10 mmol/L Tris-HCl ( p H 8.0) lmmol/L EDTA (p H 8.0) Double distilled water WdH20 ) dilution I : 10. Apparatus Beckman rotor, SW28 or equivalent Membrane (0.45 um, 500 ml) Incubation, 30°C or 37°C SW 28 Ultra-Clear Beckman Tubes Tissue Culture Plates (I O c m ) Methods 1 The day before transfection, IOcm plates of DMEM medium were inoculated with 293T cells at 10% confluence. On the day of transfection, cells were 40%~70% confluent. 2. 2 h before transfection, replace the fresh DMEM medium. 3. Mix e n v, pack, t a n s plasmids and plasmids coding for D A F or fusion protein g p 6 4 -D A F or V S V - G - D A F according to the need of the experiment according to the proportions (Table 1, Table 2). 4- Add 125ul 2m o l / L CaClundefined 2○ ul ○ - I X T E to the plasmid mixture and add d d H 20 total volume to l m l . 5- Add I m l of DNA mixture dropwise to I m l of 2 X H B S. Shake at maximum speed and allow to stand for 30 min. 6- Slowly add precipitate dropwise to the cell. 7. Incubate for 16 h. Generate DAF-modified gp64 or VSV-O pseudotyped HIV-I-derived lentiviral vectors by coexpression of gp64-DAF or VSV-ODA F-fusion proteins, incubating at 30°C instead of the standard 37°C . 8 . Remove the medium and replace with 10 to 12 m l of fresh complete medium. Virus collection, filtration, concentration 9. 48 h after transfection, collect the medium and add fresh 10~12m l medium to the cell plate. 10.0.45um membrane filtration of the collected medium. Store the culture medium at 80°C. 11. Repeat steps 8~9 daily (2~3d after the first collection of VS V-G pseudotyped virus, 4~6d after the collection of gP 64 pseudotyped virus). Because VSV-G cytotoxicity rarely produces VSV-G pseudotyped plasmid cells that survive beyond 4d, compared to gP64 low-toxicity cells that can survive one week after transfection with gp64 in the collection medium. 12. Confluence the collected medium. Ultra-clear tubes (max. 30 ml/tube) are centrifuged for 2 h at 25,000 rpm at 4°C with a S W 28 rotor. 13. Remove the upper layer of liquid and resuspend the precipitate in complete DME medium. Store the resuspended plasmid at 80°C for further analysis. reagents Cells, exponentially growing 293 or H T 1080 (human fibrosarcoma) DMEM medium (complete and serum-free) containing 10 % fetal bovine serum Human serum complement (CHM > 100; normal and heat-inactivated) Inactivate complement, incubate at 56°C for lh. Polybrene Pseudotyped H IV-I-derived lentiviral vectors ( >107 transfection units/m l ) Water bath 37°C and 56°C Oscillating mixer Methods 1- Dilute 100ul carrier with an equal amount of human normal serum complement. Heat-inactivated human serum or complete D M E M medium was used as a control. 2-Incubate the carrier-complement mixture at 37°C for l h . Vibrate for 2 s every 15 m i n . 3- After incubation, dilute the reaction solution 10-fold with serum-free DMEM. 4 . Infect H T 1080 or 293 cells with serial dilutions of viral vectors containing encoded G FP in the presence of Polybrene at 8 wow/1111. 5. 48 h after transduction, G FP positive counts were performed on culture plates containing the highest dilution of viral vector. 6. Calculate the viral titer (positive expression as G F P transduction units/m l ; G F P -T U /m l ). For more product details, please visit Aladdin Scientific website.
Plasmids encoding green fluorescent protein should be constructed.
Instrumentation
Viral titer = ( number of G F P foci X dilution factor)/volume of vector used for transduction